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Engineered chromosome regions with altered sequence composition demonstrate hierarchical large-scale folding within metaphase chromosomes.

Strukov YG, Wang Y, Belmont AS - J. Cell Biol. (2003)

Bottom Line: We engineered labeled chromosome regions with altered scaffold-associated region (SAR) sequence composition as a formal test of the radial loop and other chromosome models.Specifically, an approximately 250-nm-diam folding subunit was visualized directly within fully condensed metaphase chromosomes.Our results contradict predictions of simple radial loop models and provide the first unambiguous demonstration of a hierarchical folding subunit above the level of the 30-nm fiber within normally condensed metaphase chromosomes.

View Article: PubMed Central - PubMed

Affiliation: Dept. of Cell and Structural Biology, University of Illinois Urbana-Champaign, B107 CLSL, 601 S. Goodwin Ave., Urbana, IL 61801, USA.

ABSTRACT
Mitotic chromosome structure and DNA sequence requirements for normal chromosomal condensation remain unknown. We engineered labeled chromosome regions with altered scaffold-associated region (SAR) sequence composition as a formal test of the radial loop and other chromosome models. Chinese hamster ovary cells were isolated containing high density insertions of a transgene containing lac operator repeats and a dihydrofolate reductase gene, with or without flanking SAR sequences. Lac repressor staining provided high resolution labeling with good preservation of chromosome ultrastructure. No evidence emerged for differential targeting of SAR sequences to a chromosome axis within native chromosomes. SAR sequences distributed uniformly throughout the native chromosome cross section and chromosome regions containing a high density of SAR transgene insertions showed normal diameter and folding. Ultrastructural analysis of two different transgene insertion sites, both spanning less than the full chromatin width, clearly contradicted predictions of simple radial loop models while providing strong support for hierarchical models of chromosome architecture. Specifically, an approximately 250-nm-diam folding subunit was visualized directly within fully condensed metaphase chromosomes. Our results contradict predictions of simple radial loop models and provide the first unambiguous demonstration of a hierarchical folding subunit above the level of the 30-nm fiber within normally condensed metaphase chromosomes.

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Normal chromosome morphology over insert regions by light microscopy. In native chromosomes, the vector insert appears as a band going over the entire width of the chromosome, or as a pair of spots within normal diameter chromosome. (A) Con-1 clone showing insert across entire chromosome width. (B) Con-610 clone with single insert site. (C) dSAR-d11 clone showing two insert sites. (D) dSAR-g12 clone showing two insert sites. (column 1) Lac repressor immunostaining (green) and DAPI staining (red) of metaphase cells; (columns 2–4) metaphase chromosomes at vector integration sites: lac repressor immunostaining (green), DAPI staining (red, or grayscale). (column 5) Chromosome structure at the regions of vector insertion for cells expressing GFP–lacI–NLS fusion protein. Bars, 1 μm (white, column 1; black, columns 2–5).
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fig5: Normal chromosome morphology over insert regions by light microscopy. In native chromosomes, the vector insert appears as a band going over the entire width of the chromosome, or as a pair of spots within normal diameter chromosome. (A) Con-1 clone showing insert across entire chromosome width. (B) Con-610 clone with single insert site. (C) dSAR-d11 clone showing two insert sites. (D) dSAR-g12 clone showing two insert sites. (column 1) Lac repressor immunostaining (green) and DAPI staining (red) of metaphase cells; (columns 2–4) metaphase chromosomes at vector integration sites: lac repressor immunostaining (green), DAPI staining (red, or grayscale). (column 5) Chromosome structure at the regions of vector insertion for cells expressing GFP–lacI–NLS fusion protein. Bars, 1 μm (white, column 1; black, columns 2–5).

Mentions: At light microscopy resolution, metaphase chromosomes within intact metaphase plates had a normal chromosomal width at the insert sites (Fig. 5)Figure 5.


Engineered chromosome regions with altered sequence composition demonstrate hierarchical large-scale folding within metaphase chromosomes.

Strukov YG, Wang Y, Belmont AS - J. Cell Biol. (2003)

Normal chromosome morphology over insert regions by light microscopy. In native chromosomes, the vector insert appears as a band going over the entire width of the chromosome, or as a pair of spots within normal diameter chromosome. (A) Con-1 clone showing insert across entire chromosome width. (B) Con-610 clone with single insert site. (C) dSAR-d11 clone showing two insert sites. (D) dSAR-g12 clone showing two insert sites. (column 1) Lac repressor immunostaining (green) and DAPI staining (red) of metaphase cells; (columns 2–4) metaphase chromosomes at vector integration sites: lac repressor immunostaining (green), DAPI staining (red, or grayscale). (column 5) Chromosome structure at the regions of vector insertion for cells expressing GFP–lacI–NLS fusion protein. Bars, 1 μm (white, column 1; black, columns 2–5).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172725&req=5

fig5: Normal chromosome morphology over insert regions by light microscopy. In native chromosomes, the vector insert appears as a band going over the entire width of the chromosome, or as a pair of spots within normal diameter chromosome. (A) Con-1 clone showing insert across entire chromosome width. (B) Con-610 clone with single insert site. (C) dSAR-d11 clone showing two insert sites. (D) dSAR-g12 clone showing two insert sites. (column 1) Lac repressor immunostaining (green) and DAPI staining (red) of metaphase cells; (columns 2–4) metaphase chromosomes at vector integration sites: lac repressor immunostaining (green), DAPI staining (red, or grayscale). (column 5) Chromosome structure at the regions of vector insertion for cells expressing GFP–lacI–NLS fusion protein. Bars, 1 μm (white, column 1; black, columns 2–5).
Mentions: At light microscopy resolution, metaphase chromosomes within intact metaphase plates had a normal chromosomal width at the insert sites (Fig. 5)Figure 5.

Bottom Line: We engineered labeled chromosome regions with altered scaffold-associated region (SAR) sequence composition as a formal test of the radial loop and other chromosome models.Specifically, an approximately 250-nm-diam folding subunit was visualized directly within fully condensed metaphase chromosomes.Our results contradict predictions of simple radial loop models and provide the first unambiguous demonstration of a hierarchical folding subunit above the level of the 30-nm fiber within normally condensed metaphase chromosomes.

View Article: PubMed Central - PubMed

Affiliation: Dept. of Cell and Structural Biology, University of Illinois Urbana-Champaign, B107 CLSL, 601 S. Goodwin Ave., Urbana, IL 61801, USA.

ABSTRACT
Mitotic chromosome structure and DNA sequence requirements for normal chromosomal condensation remain unknown. We engineered labeled chromosome regions with altered scaffold-associated region (SAR) sequence composition as a formal test of the radial loop and other chromosome models. Chinese hamster ovary cells were isolated containing high density insertions of a transgene containing lac operator repeats and a dihydrofolate reductase gene, with or without flanking SAR sequences. Lac repressor staining provided high resolution labeling with good preservation of chromosome ultrastructure. No evidence emerged for differential targeting of SAR sequences to a chromosome axis within native chromosomes. SAR sequences distributed uniformly throughout the native chromosome cross section and chromosome regions containing a high density of SAR transgene insertions showed normal diameter and folding. Ultrastructural analysis of two different transgene insertion sites, both spanning less than the full chromatin width, clearly contradicted predictions of simple radial loop models while providing strong support for hierarchical models of chromosome architecture. Specifically, an approximately 250-nm-diam folding subunit was visualized directly within fully condensed metaphase chromosomes. Our results contradict predictions of simple radial loop models and provide the first unambiguous demonstration of a hierarchical folding subunit above the level of the 30-nm fiber within normally condensed metaphase chromosomes.

Show MeSH
Related in: MedlinePlus